Is DNA concentration and 260/280 ratio good indicator for further processing like whether proceed for PCR and sequencing??
In some cases i have found that even when DNA concentration and 260/280 ratio was negative, PCR and sequencing success was satisfactory.
Thanks in advance
The 260/230 ratio is used to indicate the presence of unwanted organic compounds such as Trizol, phenol, Guanidine HCL and guanidine thiocyanate. Generally acceptable 260/230 ratios are in the range of 2.0 – 2.2
The ratio of absorbance at 260 and 230 nm can be used as a secondary measure of DNA or RNA purity. In this case, a ratio between 2.0 - 2.2 is considered pure. If the ratio is lower than this expected range, it may indicate contaminants in the sample that absorb at 230nm.
Abnormal value (high or low) of 260/230 may indicate problem with a sample or with extraction procedure. This info may help
1. A low A 260/A230 ratio may be the result of:
• Carbohydrate carryover (often a problem with plants).
• Residual phenol from nucleic acid extraction.
• Residual guanidine (often used in column based kits).
• Glycogen used for precipitation.
2. A high A260/A230 ratio may be the result of:
• Making a Blank measurement on a dirty pedestal
• Using an inappropriate solution for the Blank measurement. The blank solution should be the same pH and of a similar ionic strength as the sample solution.
Example: Using water for the Blank measurement for samples dissolved in TE may result in low 260/230 ratios.
Good luck
Reference:
William W. Wilfinger, Karol Mackey, and Piotr Chomczynski,
Effect of pH and Ionic Strength on the Spectrophotometric
Assessment of Nucleic Acid Purity: BioTechniques 22:474-481
(March 1997)